IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF MASTER REGULATORS OF THE ONSET OF BERRY RIPENING IN GRAPEVINE (Vitis vinifera L.)

Grapevine is one of the most important and cultivated fruit crops in the world. Its economic importance is especially related to winemaking and the production of high-quality grape is one of the major concerns of the viticulturists. In the last years, continuous temperatures increasing have caused an anticipation of the onset of berry ripening, called veraison, modifying the physiological characteristics of grape, its final quality and consequently wine quality. To prevent these negative effects, the interpretation of the molecular mechanisms controlling this process could provide allow the development of more specific and targeted intervention strategies. To this aim, many molecular studies have been performed. One of the most important is represented by the generation of the grapevine gene expression atlas; this study showed a transcriptomic reprogramming during the vegetative-to-mature transition, suggesting the existence of key regulator genes. Further studies showed that this phase transition seem to be regulated by specific genes, defined switch genes; they are mainly transcription factors and the identification of the functions of these genes could provide important details about the molecular mechanism controlling the maturation process in grapevine. Among these transcription factors, five of them, VviNAC33, VviNAC60, VviAGL15, VviWRKY19 and VvibHLH75, have been selected for functional characterization. Their functional analysis in grapevine has been performed using stable genetic transformation and transient gene expression approaches. The application, improvement and development of these approaches has supported the functional characterization of the five selected genes. Regarding the stable transformation, to identify a standard method, 3 different protocols in 3 different cultivars, using GFP as reporter gene, have been tested. The results showed that the regeneration of transgenic somatic embryos and plants occurred only in Shiraz and Garganega cultivars using embryogenic calli as transformation material, indicating that this complex process is cultivar-dependent. Stable genetic transformation was used for the functional analysis of both VviNAC33 and VviNAC60. In a previous work, both NAC genes have been overexpressed in grapevine plants; the overexpression of VviNAC33 has altered the chlorophyll metabolism, while the overexpression of VviNAC60 has caused stunted growth and anthocyanins leaf accumulation, indicating that both genes are involved in the regulation of vegetative-to-mature transition. In this PhD project, both NAC gene have been fused with EAR motif, the strongest transcriptional repression domain in plants, and stably expressed in Garganega and Shiraz plants. The results showed that some putative target genes of both NAC transcription factors are less expressed than WT plants, indicating that EAR motif represents a good approach to study the function of a transcription factor. Regarding transient gene expression, this method was used for the functional analysis of VviAGL15, VviWRKY19 and VvibHLH75. Leaf agroinfiltration was optimized using YFP as reporter gene and tested in different cultivars by a vacuum system. The analysis of YFP transient expression showed that the fluorescence signal is especially localized in the first and second leaf from apex. VviAGL15, VviWRKY19 and VvibHLH75 have been functionally characterized using the improved leaf agroinfiltration protocol. Each transcription factor was co-expressed with YFP gene: the visualization of its expression has allowed to select only agroinfiltrated leaves. Next microarray analysis of overexpressing leaves showed that many upregulated genes are involved in processes associate with ripening, and an exhaustive molecular interpretation of these preliminary results seem to indicate that VviAGL15, VviWRKY19 and VvibHLH75 are master regulators of the onset of berry ripening, controlling many aspects of the maturation programs

DNA-free genome editing in grapevine using CRISPR/Cas9 ribonucleoprotein complexes followed by protoplast regeneration

CRISPR/Cas9 genome editing technology can overcome many limitations of traditional breeding, offering enormous potential for crop improvement and food production. Although the direct delivery of Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes to grapevine (Vitis vinifera) protoplasts has been shown before, the regeneration of edited protoplasts into whole plants has not been reported. Here, we describe an efficient approach to obtain transgene-free edited grapevine plants by the transfection and subsequent regeneration of protoplasts isolated from embryogenic callus. As proof of concept, a single-copy green fluorescent protein reporter gene (GFP) in the grapevine cultivar Thompson Seedless was targeted and knocked out by the direct delivery of RNPs to protoplasts. CRISPR/Cas9 activity, guided by two independent sgRNAs, was confirmed by the loss of GFP fluorescence. The regeneration of GFP– protoplasts into whole plants was monitored throughout development, confirming that the edited grapevine plants were comparable in morphology and growth habit to wild-type controls. We report the first highly efficient protocol for DNA-free genome editing in grapevine by the direct delivery of preassembled Cas9-sgRNA RNP complexes into protoplasts, helping to address the regulatory concerns related to genetically modified plants. This technology could encourage the application of genome editing for the genetic improvement of grapevine and other woody crop plants

Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required

The transcription factor VviNAC60 regulates senescence- and ripening-related processes in grapevine

: Grapevine (Vitis vinifera L.) is one of the most widely cultivated fruit crops because the winemaking industry has huge economic relevance worldwide. Uncovering the molecular mechanisms controlling the developmental progression of plant organs will prove essential for maintaining high-quality grapes, expressly in the context of climate change, which impairs the ripening process. Through a deep inspection of transcriptomic data, we identified VviNAC60, a member of the NAC transcription factor family, as a putative regulator of grapevine organ maturation. We explored VviNAC60 binding landscapes through DNA affinity purification followed by sequencing and compared bound genes with transcriptomics datasets from grapevine plants stably and transiently overexpressing VviNAC60 to define a set of high-confidence targets. Among these, we identified key molecular markers associated with organ senescence and fruit ripening. Physiological, metabolic, and promoter activation analyses showed that VviNAC60 induces chlorophyll degradation and anthocyanin accumulation through the up-regulation of STAY-GREEN PROTEIN 1 (VviSGR1) and VviMYBA1, respectively, with the latter being up-regulated through a VviNAC60-VviNAC03 regulatory complex. Despite sharing a closer phylogenetic relationship with senescence-related homologues to the NAC transcription factor AtNAP, VviNAC60 complemented the non-ripening(nor) mutant phenotype in tomato (Solanum lycopersicum), suggesting a dual role as an orchestrator of both ripening- and senescence-related processes. Our data support VviNAC60 as a regulator of processes initiated in the grapevine vegetative- to mature-phase organ transition and therefore as a potential target for enhancing the environmental resilience of grapevine by fine-tuning the duration of the vegetative phase

Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy

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